Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.     

Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography

Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells – especially those in tissue – are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.     

Revealing bacterial cell biology using cryo-electron tomography

Visualizing macromolecules inside bacteria at a high spatial resolution has remained a challenge owing to their small size and limited resolution of optical microscopy techniques. Recent advances in cryo-electron tomography (cryo-ET) imaging methods have revealed the spatial and temporal assemblies of many macromolecules involved in different cellular processes in bacteria at a resolution of a few nanometers in their native milieu. Specifically, the application of cryo-focused ion beam (cryo-FIB) milling to thin bacterial specimens makes them amenable for high-resolution cryo-ET data collection. In this review, we highlight recent research in three emerging areas of bacterial cell biology that have benefited from the cryo-FIB-ET technology - cytoskeletal filament assembly, intracellular organelles, and multicellularity.     

Mechanistic insight into heterogeneity of trans-plasma membrane electron transport in cancer cell types

Trans-plasma membrane electron transfer (tMPET) is a process by which reducing equivalents, either electrons or reductants like ascorbic acid, are exported to the extracellular environment by the cell. TPMET is involved in a number of physiological process and has been hypothesised to play a role in the redox regulation of cancer metabolism. Here, we use a new electrochemical assay to elucidate the ‘preference’ of cancer cells for different trans tPMET systems. This aids in proving a biochemical framework for the understanding of tPMET role, and for the development of novel tPMET-targeting therapeutics. We have delineated the mechanism of tPMET in 3 lung cancer cell models to show that the external electron transfer is orchestrated by ascorbate mediated shuttling via tPMET. In addition, the cells employ a different, non-shuttling-based mechanism based on direct electron transfer via Dcytb. Results from our investigations indicate that tPMETs are used differently, depending on the cell type. The data generated indicates that tPMETs may play a fundamental role in facilitation of energy reprogramming in malignant cells, whereby tPMETs are utilised to supply the necessary energy requirement when mitochondrial stress occurs. Our findings instruct a deeper understanding of tPMET systems, and show how different cancer cells may preferentially use distinguishable tPMET systems for cellular electron transfer processes.     

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral E^rns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic.     

Cryo electron tomography of vitrified fibroblasts: microtubule plus ends in situ

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 nm over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5–3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759–765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex.     

Stopped-Flow Fluorescence Kinetic Study of Protein Sliding and Intersegment Transfer in the Target DNA Search Process

Kinetic characterizations of protein translocation on DNA are nontrivial because the simultaneous presence of multiple different mechanisms makes it difficult to extract the information specific to a particular translocation mechanism. In this study, we have developed new approaches for the kinetic investigations of proteins' sliding and intersegment transfer (also known as “direct transfer”) in the target DNA search process. Based on the analytical expression of the mean search time for the discrete-state stochastic model, we derived analytical forms of the apparent rate constant k_app for protein-target association in systems involving competitor DNA and the intersegment transfer mechanism. Our analytical forms of k_app facilitate the experimental determination of the kinetic rate constants for intersegment transfer and sliding in the target association process. Using stopped-flow fluorescence data for the target association kinetics along with the analytical forms of k_app, we have studied the translocation of the Egr-1 zinc-finger protein in the target DNA association process. Sliding was analyzed using the DNA-length-dependent k_app data. Using the dependence of k_app on the concentration of competitor DNA, we determined the second-order rate constant for intersegment transfer. Our results indicate that a major pathway in the target association process for the Egr-1 zinc-finger protein is the one involving intersegment transfer to a nonspecific site and the subsequent sliding to the target.     

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860–4867) except that the free energy gap ΔG is used instead of the energy gap ΔE. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P⁺ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from Q^?_A to P⁺, the very slow back electron transfer from I^? to P⁺ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.     

Simple techniques for freeze clamping and for cutting and milling of frozen tissue at low temperature for the purpose of two- or three-dimensional metabolic studies in vivo

A technique is described for freeze-clamping of parenchymal tissue (e.g., liver) which causes the tissue to be rigidly fixed to an aluminium cup in the frozen state with a well-defined, reproducible orientation of the tissue as well as a minimum of morphological distortion of the major part of the sample. Furthermore, three instruments for low-temperature cutting or milling of the frozen sample for the purpose of two- or three-dimensional metabolic studies are described. The cutting and milling instruments work according to the principle of ordinary workshop machines for steel work. The frozen sample fixed in the aluminium cup may be mounted in the milling instrument and cut at the temperature of liquid nitrogen with high precision; e.g., one instrument may be adjusted to mill off tissue layers of a thickness of only 20 μm. Thermocouple readings from the frozen sample suggest that the milling process does not cause significant heating of the sample. This is further supported by the fact that the amount of labile metabolites, ATP, ADP, AMP, lactate, and pyruvate, is unaffected by the milling process.     

Reorganization of the Photosystem II Unit in Developing Thylakoids of Higher Plants after Transfer to Darkness : Changes in Chlorophyll b, Light-Harvesting Chlorophyll Protein Content, and Grana Stacking

A light-dependent reversible grana stacking-unstacking process, paralleled by a reorganization of thylakoid components, has been noticed in greening etiolated bean (Phaseolus vulgaris, var. red kidney) leaves upon transfer to darkness. The reorganization, based on biochemical and biophysical criteria, involves mainly the photosystem II (PSII) unit components: upon transfer to darkness, the light-harvesting chlorophyll protein (LHCP), its 25 kilodalton polypeptide and chlorophyll b are decreased, while the CPa and its 42 kilodalton polypeptide are increased and new PSII units of smaller size are formed. This reorganization of components occurs only in thylakoids still in the process of development and not in those present in steady state conditions.

It is proposed that this process does not reflect the turnover of the LHCP component per se, but a regulatory process operating during development, by which the ratio of light-harvesting to PSII reaction center components, determined by the environmental conditions, controls the photosynthetic rate.

     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.     

The Impact of the Invasive Alien Plant,Impatiens glandulifera,on Pollen Transfer Networks

Biological invasions are a threat to the maintenance of ecological processes, including pollination. Plant-flower visitor networks are traditionally used as a surrogated for pollination at the community level, despite they do not represent the pollination process, which takes place at the stigma of plants where pollen grains are deposited. Here we investigated whether the invasion of the alien plant Impatiens glandulifera (Balsaminaceae) affects pollen transfer at the community level. We asked whether more alien pollen is deposited on the stigmas of plants on invaded sites, whether deposition is affected by stigma type (dry, semidry and wet) and whether the invasion of I. glandulifera changes the structure of the resulting pollen transfer networks. We sampled stigmas of plants on 10 sites invaded by I. glandulifera (hereafter, balsam) and 10 non-invaded control sites. All 20 networks had interactions with balsam pollen, although significantly more balsam pollen was found on plants with dry stigmas in invaded areas. Balsam pollen deposition was restricted to a small subset of plant species, which is surprising because pollinators are known to carry high loads of balsam pollen. Balsam invasion did not affect the loading of native pollen, nor did it affect pollen transfer network properties; networks were modular and poorly nested, both of which are likely to be related to the specificity of pollen transfer interactions. Our results indicate that pollination networks become more specialized when moving from the flower visitation to the level of pollen transfer networks. Therefore, caution is needed when inferring pollination from patterns of insect visitation or insect pollen loads as the relationship between these and pollen deposition is not straightforward.     

Are Protein Domains Modules of Lateral Genetic Transfer?

Background

In prokaryotes and some eukaryotes, genetic material can be transferred laterally among unrelated lineages and recombined into new host genomes, providing metabolic and physiological novelty. Although the process is usually framed in terms of gene sharing (e.g. lateral gene transfer, LGT), there is little reason to imagine that the units of transfer and recombination correspond to entire, intact genes. Proteins often consist of one or more spatially compact structural regions (domains) which may fold autonomously and which, singly or in combination, confer the protein''s specific functions. As LGT is frequent in strongly selective environments and natural selection is based on function, we hypothesized that domains might also serve as modules of genetic transfer, i.e. that regions of DNA that are transferred and recombined between lineages might encode intact structural domains of proteins.

Methodology/Principal Findings

We selected 1,462 orthologous gene sets representing 144 prokaryotic genomes, and applied a rigorous two-stage approach to identify recombination breakpoints within these sequences. Recombination breakpoints are very significantly over-represented in gene sets within which protein domain-encoding regions have been annotated. Within these gene sets, breakpoints significantly avoid the domain-encoding regions (domons), except where these regions constitute most of the sequence length. Recombination breakpoints that fall within longer domons are distributed uniformly at random, but those that fall within shorter domons may show a slight tendency to avoid the domon midpoint. As we find no evidence for differential selection against nucleotide substitutions following the recombination event, any bias against disruption of domains must be a consequence of the recombination event per se.

Conclusions/Significance

This is the first systematic study relating the units of LGT to structural features at the protein level. Many genes have been interrupted by recombination following inter-lineage genetic transfer, during which the regions within these genes that encode protein domains have not been preferentially preserved intact. Protein domains are units of function, but domons are not modules of transfer and recombination. Our results demonstrate that LGT can remodel even the most functionally conservative modules within genomes.     

Conformational changes in photosynthetic pigment proteins on thylakoid membranes can lead to fast non-photochemical quenching in cyanobacteria

A high non-photochemical quenching (NPQ) appeared below the phase transition temperature when Microcystis aeruginosa PCC7806 cells were exposed to saturated light for a short time. This suggested that a component of NPQ, independent from state transition or photo-inhibition, had been generated in the PSII complex; this was a fast component responding to high intensity light. Glutaraldehyde (GA), commonly used to stabilize membrane protein conformations, resulted in more energy transfer to PSII reaction centers, affecting the energy absorption and dissipation process rather than the transfer process of phycobilisome (PBS). In comparison experiments with and without GA, the rapid light curves (RLCs) and fluorescence induction dynamics of the fast phase showed that excess excitation energy was dissipated by conformational change in the photosynthetic pigment proteins on the thylakoid membrane (PPPTM). Based on deconvolution of NPQ relaxation kinetics, we concluded that the fast quenching component (NPQf) was closely related to PPPTM conformational change, as it accounted for as much as 39.42% of the total NPQ. We hypothesize therefore, that NPQf induced by PPPTM conformation is an important adaptation mechanism for Microcystis blooms under high-intensity light during summer and autumn.     

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

Structure of a photosystem I-ferredoxin complex from a marine cyanobacterium provides insights into far-red light photoacclimation

Far-red light photoacclimation exhibited by some cyanobacteria allows these organisms to use the far-red region of the solar spectrum (700–800 nm) for photosynthesis. Part of this process includes the replacement of six photosystem I (PSI) subunits with isoforms that confer the binding of chlorophyll (Chl) f molecules that absorb far-red light (FRL). However, the exact sites at which Chl f molecules are bound are still challenging to determine. To aid in the identification of Chl f-binding sites, we solved the cryo-EM structure of PSI from far-red light-acclimated cells of the cyanobacterium Synechococcus sp. PCC 7335. We identified six sites that bind Chl f with high specificity and three additional sites that are likely to bind Chl f at lower specificity. All of these binding sites are in the core-antenna regions of PSI, and Chl f was not observed among the electron transfer cofactors. This structural analysis also reveals both conserved and nonconserved Chl f-binding sites, the latter of which exemplify the diversity in FRL-PSI among species. We found that the FRL–PSI structure also contains a bound soluble ferredoxin, PetF1, at low occupancy, which suggests that ferredoxin binds less transiently than expected according to the canonical view of ferredoxin-binding to facilitate electron transfer. We suggest that this may result from structural changes in FRL-PSI that occur specifically during FRL photoacclimation.     

Cholesterol efflux from larval Manduca sexta fat body in vitro: high-density lipophorin as the acceptor

The objective of this study was to characterize the transfer of cholesterol from Manduca sexta larvae fat body to high-density lipophorin. [³H]-Cholesterol-labeled fat body was incubated with lipophorin under different conditions and cholesterol transfer was determined. Transfer rate exhibited a hyperbolic dependency on lipophorin concentration with an apparent K_m of 3.6 mg/ml, which is consistent with either an aqueous diffusion mechanism of cholesterol transfer or a receptor-mediated process. Several results, including the high K_m, the high activation energy, and the lack of Ca²⁺ dependence favor aqueous diffusion model. In addition, anti-lipid transfer particle antibodies had only a small inhibitory effect, suggesting it is not involved in cholesterol transfer. However, the transfer was inhibited in the presence of suramin, which would be consistent with a receptor-mediated process. The effects of suramin may be complex because it can change membrane properties when bound to the lipophorin receptor and affect the rate of cholesterol desorption. The preponderance of data suggests that the export of cholesterol from fat body to lipophorin follows a simple aqueous diffusion pathway. Although we cannot completely exclude some contribution from a receptor-mediated pathway, it seems that if such a pathway were present, it represents a minor route.     

Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.     

Complexity, connectivity, and duplicability as barriers to lateral gene transfer

Background

Lateral gene transfer is a major force in microbial evolution and a great source of genetic innovation in prokaryotes. Protein complexity has been claimed to be a barrier for gene transfer, due to either the inability of a new gene's encoded protein to become a subunit of an existing complex (lack of positive selection), or from a harmful effect exerted by the newcomer on native protein assemblages (negative selection).

Results

We tested these scenarios using data from the model prokaryote Escherichia coli. Surprisingly, the data did not support an inverse link between membership in protein complexes and gene transfer. As the complexity hypothesis, in its strictest sense, seemed valid only to essential complexes, we broadened its scope to include connectivity in general. Transferred genes are found to be less involved in protein-protein interactions, outside stable complexes, and this is especially true for genes recently transferred to the E. coli genome. Thus, subsequent to transfer, new genes probably integrate slowly into existing protein-interaction networks. We show that a low duplicability of a gene is linked to a lower chance of being horizontally transferred. Notably, many essential genes in E. coli are conserved as singletons across multiple related genomes, have high connectivity and a highly vertical phylogenetic signal.

Conclusion

High complexity and connectivity generally do not impede gene transfer. However, essential genes that exhibit low duplicability and high connectivity do exhibit mostly vertical descent.